Tyrosine kinase-mediated signal transduction pathway in smooth muscle activated by melatonin

Abstract

The characteristics of melatonin receptors and mechanism of melatonin action on the quail caecum were investigated. In the crude caecal membrane preparation, saturation, kinetic and competition study revealed a single population of specific, high affinity, and low-density binding sites for 2[125I]iodomelatonin (Kd = 24.6 B 1.1 pmol/l; Bmax = 2.0 B 0.1 fmol/mg; n = 7). The biochemical and pharmacological characteristics of 2[125I]iodomelatonin binding are similar to the ML1 high-affinity melatonin receptors reported. Using the caecal ring as our study model, we showed that melatonin was able to potentiate K+ (30 mM)-induced contraction in a dose dependent manner (EC50 = 1.38 ÌM). Luzindol and 4-phenyl–2-propionamidotetraline (4-P-PDOT), two antagonists of ML1 receptor attenuated the melatonin constrictor responses with Ki = 6.4 ÌM and Ki = 3.7 ÌM, respectively. Epidermal growth factor (EGF) exerted similar potentiation as that of melatonin on the K+-predepolarized quail caecum. Genistein (40 ÌM) and erbstatin (10 Ìg/ml), two tyrosine kinases blockers, completely inhibited the contractile responses of both of the agonists studied. In contrast to melatonin and EGF, agents such as epinephrine (1 ÌM) and dibutyrylcAMP (1 mM), which are known to enhance the level of the intracellular cAMP thereby initiating protein kinase A (PKA), completely inhibited the K+-induced contraction. Moreover, an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate, failed to modify muscular responses of quail caecum to K+. The effect of melatonin in caecum rings was accompanied by an increased intracellular Ca2+ concentration ([Ca2+]i), as estimated by fura-2. The melatonin-promoted rise in [Ca2+]i, just as the K+-induced [Ca2+]I enhancement, seems to be the result of an activation of potential-dependent Ca2+ channels by both K+ and melatonin, since a specific blocker of L-type Ca2+ channels, nifedipine (1 ÌM), inhibited the contractile increments. Our results indicate that a high-affinity melatonin receptor-couple pathway of contraction is present in the quail caecum visceral smooth muscle. We also suggest that melatonin potentiates the K+-induced caecum contraction by activating ML1 receptors, which mobilize tyrosine kinases, but not PKA or PKC. The melatonin stimulation on smooth muscle may mediate through a tyrosyl phosphorylation of subunits of L-type Ca2+ channel.